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Cytoskeleton morphology plays a key role in regulating cell mechanics. Particularly, cellular mechanical properties are directly regulated by the highly cross-linked and dynamic cytoskeletal structure of F-actin and microtubules presented in the cytoplasm. Although great efforts have been devoted to investigating the qualitative relation between the cellular cytoskeleton state and cell mechanical properties, comprehensive quantification results of how the states of F-actin and microtubules affect mechanical behavior are still lacking. In this study, the effect of both F-actin and microtubules morphology on cellular mechanical properties was quantified using atomic force microscope indentation experiments together with the proposed image recognition-based cytoskeleton quantification approach. Young’s modulus and diffusion coefficient of NIH/3T3 cells with different cytoskeleton states were quantified at different length scales. It was found that the living NIH/3T3 cells sense and adapt to the F-actin and microtubules states: both the cellular elasticity and poroelasticity are closely correlated to the depolymerization degree of F-actin and microtubules at all measured indentation depths. Moreover, the significance of the quantitative effects of F-actin and microtubules in affecting cellular mechanical behavior is depth-dependent. 

Living cells sense and respond to their extracellular environment. Their contact guidance is affected by the underlying substrate morphology. Previous studies of the effect of substrate pattern on the mechanical behavior of living cells were only limited to the quantification of the cellular elasticity. However, how the length and time scales of the cellular mechanical properties are affected by the patterned substrates have yet to be studied. In this study, the effect of the substrate morphology on the biomechanical behavior of living cells was thoroughly investigated using indentation-based atomic force microscopy. The results showed that the cellular biomechanical behavior was affected by the substrate morphology significantly. The elasticity and viscosity of the cells on the patterned PDMS substrates were much lower compared to those cultured on flat PDMS. The poroelastic diffusion coefficient of the cells was higher on the patterned PDMS substrates, specifically on the substrate with 2D pitches. In addition, fluorescence images showed that the substrate topography directly affects the cell cytoskeleton morphology. Together, the results suggested that cell mechanical behavior and morphology can be controlled using substrates with properly designed topography.